Predictive significance of Charcot‐Leyden crystal structures for nasal polyp recurrence

Abstract Background Charcot‐Leyden crystals (CLCs) are recognized to be classic hallmarks of eosinophilic inflammation. Both protein and mRNA levels of CLC in nasal secretions and nasal brushing samples have been associated with nasal polyp recurrence. However, whether the crystalline CLC structures in nasal tissue could serve as an effective biomarker to predict polyp recurrence remains unclear. Methods A total of 110 patients with chronic rhinosinusitis with nasal polyps (CRSwNP) completing the postoperative follow‐up over a period of 24 months were recruited. Hematoxylin and eosin staining was employed for CLCs identification. The predictive factors for polyp recurrence were determined by binary logistic regression analysis. Results Thirty three (30.00%) patients developed recurrence during a 24‐month postoperative follow‐up, in which 84.85% (28/33) patients had crystalline CLC structures. Logistic regression analysis showed that crystalline CLC structure in nasal tissues is predictive of polyp recurrence. Youden index demonstrated crystalline CLC structure higher than 1 per high power field can predict postoperative polyp recurrence with 84.80% sensitivity and 98.70% specificity. Conclusions The crystalline CLC structures in nasal tissues may serve as an easy‐counting and promising biomarker to predict CRSwNP recurrence.

To the editor, Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous and challenging inflammatory airway disease involving noneosinophilic and eosinophilic endotypes. In fact, 98.5% eosinophilic CRSwNP (Eos CRSwNP) patients were recurrent after undergone endoscopic sinus surgery (ESS). 1 Therefore, the identification of predictors of recurrence for Eos CRSwNP is crucial for better disease control. The local tissue eosinophilia has been proven to be the most important predictor. 2 However, there are no standard methods for evaluating tissue eosinophilia due to uneven distribution and diversity in geographic conditions. 2 4 Recent studies have indicated that CLC mRNA and protein levels in nasal brushing/secretions/tissues detected by ELISA and PCR were used as predictive indicators for recurrent CRSwNP and glucocorticoid sensitivity. [5][6][7] Since only crystalline CLCs, but not soluble CLC/Gal-10 protein, drive type 2 immunity, allergy and neutrophilic inflammation, 8,9 the predictive value of functional CLCs for nasal polyp (NP) relapse should be evaluated. However, little is known regarding whether crystalline CLC structures can be utilized to predict NP recurrence.
This was a retrospective study using data collected from patients with bilateral NPs who had undergone ESS at The Third Affiliated Hospital of Sun Yat-sen University from January 2016 to December 2016. The patients treated with oral/nasal glucocorticoids, immunomodulatory agents, or antibiotics with 1 month before enrollment were exclude, and patients with fungal sinusitis, cystic fibrosis, allergic fungal rhinosinusitis, or primary ciliary dyskinesia were excluded. Eosinophilic CRSwNP was defined when eosinophil percentage >10% of the total infiltrating cells as previously described. 10 The current study, through the post-operative follow-up of CRSwNP subjects are shown in Table 1. We found that patients from the recurrent group had increased comorbid rate of asthma (p = 0.015) compared with recurrent-free group. In addition, the percentage of male gender is 84.85% in the recurrent group, which is much higher than that in recurrent-free group (57.14%) (p = 0.005). Moreover, CLCs count (p < 0.001) and eosinophils count (p < 0.001) in nasal tissues and both percentage (p = 0.001) and count number (p < 0.001) of eosinophils in peripheral blood (PB) were significantly evaluated in recurrent group compared with recurrent-free group.
To evaluate the presence of CLCs in NP biopsies, H&E staining was performed. The bipyramidal crystalline CLC structures were present in 29 out of 110 (26.36%) patients with CRSwNP, and present in 64.44% patients with Eos CRSwNP ( Figure 1A-C). The presence of CLCs was also found in the diseased ethmoid mucosa, although the positive percentage is much lower than that in NPs (data not shown), which suggesting that there is eosinophildominated CRS in addition to eosinophil-dominated NPs. We did not find any CLCs in Non-Eos CRSwNP and control groups ( Figure 1C). In addition, our data showed that CRSwNP patients with CLCs account for 84.85% (28/33) of recurrent group, whereas CRSwNP patients without CLCs account for 15.15% (5/33) of recurrent group (Table 1). As expected, patients with Eos CRSwNP account for 87.88% (29/33) of recurrent group, whereas patients with Non-Eos CRSwNP occupy 12.12% (4/33) of recurrent group.
These results suggest CLCs, similar to eosinophils, are closely associated with NPs recurrence.
The logistic regression analysis was employed to determine the specific factors associated with polyp recurrence. Univariable parameters were based on between-group comparison analysis. The analysis demonstrated that tissue CLCs count (odds ratio = 2.203,   were shown in Figure 1D.

| CONCLUSION
The crystalline CLC structures visualized by histomorphology in NPs are more than markers of eosinophilic inflammation, but an objective and promising predictor for NP recurrence. The crystalline CLC structures may serve as a potential target for CRSwNP treatment strategy, particularly Eos CRSwNP.